Multi-level specific targeting of cancer cells

ABSTRACT

A compound comprising, in combination: a cell surface binding ligand or internalizing factor, such as an IL-13Rα2 binding ligand; at least one effector molecule (e.g., one, two, three or more effector molecules); optionally but preferably, a cytosol localization element covalently coupled between said binding ligand and said at least one effector molecule; and a subcellular compartment localization signal element covalently coupled between said binding ligand and said at least one effector molecule (and preferably with said cytosol localization element between said binding ligand and said subcellular compartment localization signal element). Methods of using such compounds and formulations containing the same are also described.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/324,952, filed Apr. 16, 2010, the disclosure ofwhich is incorporated by reference herein in its entirety.

GOVERNMENT FUNDING

This invention was made with United States government support undergrant number RO1 CA 74145 from the National Institutes of Health. TheUnited States government has certain rights to this invention.

FIELD OF THE INVENTION

The present invention concerns methods and constructs for deliveringcompounds of interest to cells, particularly cells that express IL-13receptors.

BACKGROUND OF THE INVENTION

Molecular targeting of cancer cells is achieved (a) specifically throughthe use of ligands/antibodies against tumor-associated or tumor-specificreceptors, and (b) non-specifically using plasma membrane permeableagents targeting activated/over-expressed intracellular elements, suchas the oncogenes. In the field of non-viral gene therapy of cancer thatemploys recombinant proteins, the inventors have pioneered the use ofproteinaceous vectors for the targeted intracellular transport ofproteins/non-proteinaceous compounds (1-3). Some bacterial toxins, suchas Pseudomonas exotoxin A (PE) or Diphtheria toxin (DT), possess anability to exit the endocytic compartment after being internalized inthe process of receptor-mediated internalization and beingproteolytically activated by a calcium-dependent serine endoprotease,furin (4-7). This “get cleaved and exit endocytic compartment” abilityis possible due to the presence of a specialized domain of PE, domain II(abbreviated here D2) (8; 9).

Previously, the inventors have exploited PE translocation ability totraffic other, non-PE, or repeated PE peptide sequences into the cellcytosol (1). This was achieved by incorporating non-PE peptides or anadditional catalytic domain III of PE within dispensable domain lb. Thisdomain is downstream of both furin cleavage site and a cleavage-createdN-terminal sequence important for initiation/conduct of the C-terminalportion of the toxin (portion of domain 2 and domain 3) endocyticvesicles exit. The inventors demonstrated for the first time that inthis manner, PE can serve as a vector for intra-cytosolic delivery ofvarious proteins (1).

Most anti-cancer therapeutics have defined targets such as oncogenes,enzymes or DNA, all of which are localized to distinct intra-cellularcompartments like nucleus, mitochondria or cytosol. GBM is a high-gradeastrocytoma representing the most common form of primary brain tumors.The successful treatment of patients with GBM is still a major challengeand the median survival rate is 14.5 months after diagnosis (12).Several factors specific to GBM have been uncovered in recent years(13-16). For example, a tri-molecular signature of GBM has beendocumented that includes IL-13Rα2, EphA2 receptor and a fos-relatedantigen 1 (Fra-1) (17). All three factors belonging to the signature aresuitable for therapeutic targeting of GBM (18). IL-13Rα2 is expressedin >75% of GBM tumor specimens (19; 20) and is characterized as acancer/testes like antigen (21). IL-13Rα2 is believed to act as a decoyreceptor (22). However, it has been shown that IL-13 ligand binds toIL13Rα2 receptor and is internalized through receptor mediatedendocytosis (23; 24). Thus, drugs attached to the IL-13 ligand can beinternalized and delivered specifically inside the glioma cells.

SUMMARY OF THE INVENTION

A first aspect of the invention is a compound comprising, incombination: a cell surface binding ligand or internalizing factor, suchas an IL-13Rα2 binding ligand; at least one effector molecule (e.g.,one, two, three or more effector molecules); optionally but preferably,a cytosol localization element covalently coupled between said bindingligand and said at least one effector molecule; and a subcellularcompartment localization signal element covalently coupled between saidbinding ligand and said at least one effector molecule (and preferablywith said cytosol localization element between said binding ligand andsaid subcellular compartment localization signal element).

In some embodiments, the compound has the formula, from N terminus to Cterminus, selected from the group consisting of: A-B-C-D-E; E-D-C-B-A;A-B-D-C-E; and E-C-D-B-A, wherein: A is an internalizing factor orbinding element such as an IL-13Rα2 binding ligand; B is the cytosollocalization element; C is the subcellular compartment localizationsignal element; D is present or absent and when present a first effectormolecule; and E is present or absent and when present is a secondeffector molecule. As will be appreciated, additional effector molecules(e.g., three or more effector molecules) can be included if so desired.

In some embodiments, the compound is a fusion protein or covalentconjugate.

In some embodiments, each of A, B, and C, and optionally D and E, is apeptide.

In some embodiments, the cytosol localization element is a Pseudomonasor diphtheria toxin translocation domain, such as a Pseudomonas exotoxinA D2 segment.

In some embodiments, the subcellular compartment localization signalelement is a nuclear localization element or a lysosomal localizationelement, such as an SV40 T antigen nuclear localization signal.

In some embodiments, wherein said IL-13Rα2 binding ligand is IL-13, amutant of IL-13, or an IL-13Rα2 binding fragment thereof.

A further aspect of the invention is a nucleic acid that encodes acompound as described above, along with host cells that contain andexpress the same.

A further aspect of the invention is a method of treating and/ordetecting cancer in a subject in need thereof, comprising administeringsaid subject a compound as described herein in a treatment and/ordetection effective amount. The cancer may be, for example, breastcancer, bladder cancer, pancreatic cancer, colorectal cancer, head andneck cancer, thyroid cancer, prostate cancer, and gliomas such asglioblastoma multiforme.

A further aspect of the invention is a method of detecting IL-13Rα2expressing cells, comprising administering a compound as describedherein to a cell or group of cells in vitro or in vivo, and detecting adetectable group coupled to said compound.

A further aspect of the invention is a method of delivering at least oneeffector molecule (e.g., a detectable group or a therapeutic group) to asubcellular compartment of a cell of interest, comprising: contacting acompound as described herein including at least one effector molecule(e.g., as either D or E) to a cell of interest (e.g., a eukaryotic cell,in vitro or in vivo) under conditions in which said compound isinternalized therein and said effector molecule is delivered to saidsubcellular compartment (e.g., the nucleus). In some embodiments, thecompound further comprises an additional effector molecule (e.g., aseither D or E). In some embodiments, the additional effector molecule isdelivered to the cytosol of the cell of interest (e.g., wherein saidcompound is of the formula A-B-D-C-E or E-C-D-B-A). The method is usefulfor research purposes (e.g., labeling subcellular compartments), and forthe methods of diagnosis and treatment described herein.

A further aspect of the invention is the use of a compound as describedherein for carrying out a method as described herein, and/or for thepreparation of a medicament as described herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

The disclosures of all United States patents cited herein are to beincorporated herein by reference in their entirety.

A. DEFINITIONS

“Capping group” as used herein includes, but is not limited to, acetyl,benzoyl, formyl, trifluoroacetyl, benzyloxycarbonyl,tert-butyloxycarbonyl, biphenylylisopropyloxycarbonyl, triphenylmethyl,o-nitrobenzenesulfenyl, and diphenylphosphinyl. The capping groups mayconsist of such groups as R¹⁰CO—, R¹⁰—O—CO—, R¹⁰—PO—, R¹⁰—SO₂— andarylalkyl-; where R¹⁰ is selected from the group consisting of H, alkyl,alkenyl, alkynyl, aryl, and arylalkyl.

“Alkyl,” as used herein, refers to a straight or branched chainhydrocarbon containing from 1 to 10 carbon atoms. Representativeexamples of alkyl include, but are not limited to, methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl,n-decyl, and the like. “Loweralkyl” as used herein, is a subset of alkyland refers to a straight or branched chain hydrocarbon group containingfrom 1 to 4 carbon atoms. Representative examples of lower alkylinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, tert-butyl, and the like.

“Alkenyl,” as used herein, refers to a straight or branched chainhydrocarbon containing from 2 to 10 carbons and containing at least onecarbon-carbon double bond formed by the removal of two hydrogens.Representative examples of “alkenyl” include, but are not limited to,ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl,5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl and the like.“Lower alkenyl” as used herein, is a subset of alkenyl and refers to astraight or branched chain hydrocarbon group containing from 2 to 4carbon atoms.

“Alkynyl,” as used herein, refers to a straight or branched chainhydrocarbon group containing from 2 to 10 carbon atoms and containing atleast one carbon-carbon triple bond. Representative examples of alkynylinclude, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl,3-butynyl, 2-pentynyl, 1-butynyl and the like. “Lower alkynyl” as usedherein, is a subset of alkyl and refers to a straight or branched chainhydrocarbon group containing from 2 to 4 carbon atoms.

The alkyl, alkenyl, and alkynyl groups of the invention can besubstituted or unsubstituted and are either unless otherwise specified.When substituted the alkyl, alkenyl or alkynyl groups of the inventioncan be substituted with 1, 2, 3, 4, or 5 or more substituentsindependently selected from alkenyl, alkenyloxy, alkoxy, alkoxyalkoxy,alkoxycarbonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alkylsulfinyl,alkylsulfonyl, alkylthio, alkynyl, aryl, azido, arylalkoxy, arylalkyl,aryloxy, carboxy, cyano, formyl, halogen, haloalkyl, haloalkoxy,hydroxy, hydroxyalkyl, mercapto, nitro, sulfamyl, sulfo, sulfonate,

“Aryl” as used herein, refers to a monocyclic carbocyclic ring system ora bicyclic carbocyclic fused ring system having one or more aromaticrings. Representative examples of aryl include, azulenyl, indanyl,indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.

The aryl groups of this invention can be substituted with 1, 2, 3, 4, or5 or more substituents independently selected from alkenyl, alkenyloxy,alkoxy, alkoxyalkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl,alkylcarbonyloxy, alkylsulfinyl, alkylsulfonyl, alkylthio, alkynyl,aryl, azido, arylalkoxy, arylalkyl, aryloxy, carboxy, cyano, formyl,halogen, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, mercapto, nitro,sulfamyl, sulfo, sulfonate,

“Arylalkyl,” as used herein, refers to an aryl group, as defined herein,appended to the parent molecular moiety through an alkenyl group, asdefined herein. Representative examples of arylalkenyl include, but arenot limited to, 2-phenylethenyl, 3-phenylpropen-2-yl,2-naphth-2-ylethenyl, and the like, which may be substituted orunsubstituted as noted above.

“IL13” or “IL-13” as used herein refers to interleukin-13, which is apleiotropic cytokine. IL-13 has approximately 30% sequence identity withIL4 and exhibits IL4-like activities on monocytes/macrophages and humanB cells (Minty et al. (1993) Nature 362:248; McKenzie et al. (1987)Proc. Natl. Acad. Sci. USA 90:3735). In particular, IL-13 appears to bea potent regulator of inflammatory and immune responses. IL-13 canup-regulate the monocyte/macrophage expression of CD23 and MHC class Iand class II antigens, down-regulate the expression of Fc.gamma, andinhibit antibody-dependent cytotoxicity. IL-13 can also inhibit nitricoxide production as well as the expression of pro-inflammatory cytokines(e.g., IL-1, IL-6, IL-8, IL-10 and IL-12) and chemokines (MIP-1, MCP),but enhance the production of IL-1.

“Recombinant” nucleic acid as used herein refers to a nucleic acidproduced by combining two or more nucleic acid sequences from differentsources, e.g., by use of molecular biology techniques, to form a newnucleic acid, e.g., a “heterologous” nucleic acid. The recombinantnucleic acid may be provided in the form of a “vector” or “deliveryvector” in order to transform or transfect cells to contain the newnucleic acid. As used herein, a “vector” or “delivery vector” can be aviral or non-viral vector that is used to deliver a nucleic acid to acell, tissue or subject.

A “recombinant” protein is a protein produced by a recombinant nucleicacid. The nucleic acid may or may not be inserted into the genome of ahost cell. The nucleic acid may exist, e.g., in plasmid form in a hostcell. Alternatively, the recombinant protein may be produced by in vitrotranslation of the recombinant nucleic acid.

An “isolated” protein or polypeptide means a protein or polypeptide thatis separated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other proteins or nucleicacids commonly found associated with the protein. As used herein, the“isolated” protein or polypeptide is at least about 25%, 30%, 40%, 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more pure (w/w).

“Subjects” as used herein are generally human subjects and includes, butis not limited to, cancer patients. The subjects may be male or femaleand may be of any race or ethnicity, including, but not limited to,Caucasian, African-American, African, Asian, Hispanic, Indian, etc. Thesubjects may be of any age, including newborn, neonate, infant, child,adolescent, adult, and geriatric. Subjects may also include animalsubjects, particularly mammalian subjects such as canines, felines,bovines, caprines, equines, ovines, porcines, rodents (e.g. rats andmice), lagomorphs, primates (including non-human primates), etc.,screened for veterinary medicine or pharmaceutical drug developmentpurposes.

“Cancer” or “cancers” that can be detected and/or treated by thecompounds, compositions and methods described herein include, but arenot limited to, breast cancer, bladder cancer, pancreatic cancer,colorectal cancer, head and neck cancer, thyroid cancer, prostatecancer, and brain cancer such as gliomas (e.g., GBM), etc.

“Effector molecule” as used herein includes therapeutic agents,detectable groups, targeting ligands, and delivery vehicles (e.g.,antibodies, lipids, liposomes). See, e.g., U.S. Pat. No. 6,630,576.

“Therapeutic agent” as used herein may be any therapeutic agentincluding, but not limited to, genetic materials or agents,radionuclides, chemotherapeutic agents, and cytotoxic agents (See, e.g.,U.S. Pat. No. 6,949,245 to Sliwkowski), and amphipathic antimicrobialpeptides. Other exemplary therapeutic agents include, but are notlimited to, radiopharmaceuticals, including, but not limited to augerelectrons, chemotherapeutics, and photosensitizers.

“Radionuclide” as described herein includes, but is not limited to,²²⁷Ac, ²¹¹At, ¹³¹Ba, ⁷⁷Br, ¹⁰⁹Cd, ⁵¹Cr, ⁶⁷Cu, ¹⁶⁵Dy, ¹⁵⁵Eu, ¹⁵³Gd,¹⁹⁸Au, ¹⁶⁶Ho, ^(113m)In, ^(115m)In, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁹Ir, ¹⁹¹Ir,¹⁹²Ir, ¹⁹⁴Ir, ⁵²Fe, ⁵⁵Fe, ⁵⁹Fe, ¹⁷⁷Lu, ¹⁰⁹Pd, ³²P, ²²⁶Ra, ¹⁸⁶Re, ¹⁸⁸Re,¹⁵³Sm, ⁴⁶Sc, ⁴⁷Sc, ⁷²Se, ⁷⁵Se, ¹⁰⁵Ag, ⁸⁹Sr, ³⁵S, ¹⁷⁷Ta, ¹¹⁷mSn, ¹²¹Sn,¹⁶⁶Yb, ¹⁶⁹Yb, ⁹⁰Y, ²¹²Bi, ¹¹⁹Sb, ¹⁹⁷Hg, ⁹⁷Ru, ¹⁰⁰Pd, ^(101m)Rh, and²¹²Pb.

“Chemotherapeutic agent” as used herein includes, but is not limited to,methotrexate, daunomycin, mitomycin C, cisplatin, vincristine,epirubicin, fluorouracil, verapamil, cyclophosphamide, cytosinearabinoside, aminopterin, bleomycin, mitomycin C, democolcine,etoposide, mithramycin, chlorambucil, melphalan, daunorubicin,doxorubicin, tamosifen, paclitaxel, vincristin, vinblastine,camptothecin, actinomycin D, and cytarabine. Other examples are found inU.S. Patent Application Publication 2006/0121539 (Debinski et al.),which is incorporated by reference herein in its entirety.

“Cytotoxic agent” or “toxic agent” as used herein includes, but is notlimited to, maytansinoids and maytansinoid analogs, taxoids, CC-1065 andCC-1065 analogs, dolastatin and dolastatin analogs, ricin (or moreparticularly the ricin A chain), aclacinomycin, Diphtheria toxin,Monensin, Verrucarin A, Abrin, Tricothecenes, and Pseudomonas exotoxinA, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, anti-mitotic agents, such as the vincaalkaloids (e.g., vincristine and vinblastine), colchicin,anthracyclines, such as doxorubicin and daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, and 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU),lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II)(DDP)), and antibiotics, including, but not limited to, dactinomycin(formerly actinomycin), bleomycin, mithramycin, calicheamicin, andanthramycin (AMC)).

In some embodiments, cytotoxic agents include toxins such as Pseudomonasexotoxin, ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,etc. See, e.g., U.S. Pat. No. 7,517,964. In some embodiments,Pseudomonas exotoxin or a Diphtheria toxin are preferred. See U.S. Pat.No. 5,328,984 to Pastan et al. and U.S. Pat. No. 6,296,843 to Debinski,which are each incorporated by reference herein in its entirety.Pseudomonas exotoxins can include, but are not limited to, Pseudomonasexotoxin A (PE). The Pseudomonas exotoxin can be modified such that itsubstantially lacks domain Ia, and in some embodiments Pseudomonasexotoxins include PE38QQR and PE4E. Diphtheria toxins can include DT390,a diphtheria toxin in which the native binding domain is eliminated. Itwill be appreciated that in various embodiments, the therapeutic agentscan be attached to, e.g., the amino terminus or the carboxyl terminus.

“Amphipathic antimicrobial peptide” as used herein includes amphipathicpeptides that induce apoptosis of cancer cells, presumably through theirability to depolarize mitochondrial membranes. K. Rege et al., CancerRes. 67, 6368 (Jul. 1, 2007). Such peptides are, in general, from 10, 12or 13 to 20, 30 or 40 amino acids in length, or more, and typically havean amphipathic alpha-helical structure. Examples include, but are notlimited to, (KLAKLAK)₂ (SEQ ID NO: 60); (KLAKKLA)₂ (SEQ ID NO: 61)(KAAKKAA)₂ (SEQ ID NO: 62) and (KLGKKLG)₂ (SEQ ID NO: 63) See, e.g.,Ruoslahti et al., US Patent Application 20010046498 (Nov. 29, 2001).

“Detectable group” or “label” as used herein includes, but is notlimited to, radiolabels (e.g., ³⁵S, ¹²⁵I, ³²P, ³H, ¹⁴C, ¹³¹I), enzymelabels (e.g., horseradish peroxidase, alkaline phosphatase), gold beads,chemiluminescence labels, ligands (e.g., biotin, digoxin) and/orfluorescence labels (e.g., rhodamine, phycoerythrin, fluorescein,fluorescent proteins), a fluorescent protein including, but not limitedto, a green fluorescent protein or one of its many modified forms, anucleic acid segment in accordance with known techniques, and energyabsorbing and energy emitting agents. Thus “label” or “detectable group”as used herein may be any suitable label or detectable group detectableby spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means including but not limited tobiotin, fluorophores, antigens, porphyrins, and radioactive isotopes.Labels useful in the present invention include biotin for staining withlabeled avidin or streptavidin conjugate, magnetic beads (e.g.,Dynabeads™), fluorescent dyes (e.g., fluorescein,fluorescein-isothiocyanate [FITC], Texas red, rhodamine, greenfluorescent protein, enhanced green fluorescent protein, lissamine,phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX [Amersham], SyBRGreen I & II [Molecular Probes], and the like), radiolabels (e.g., ³H,³⁵S, ¹⁴C, or ³²P), enzymes (e.g., hydrolases, particularly phosphatasessuch as alkaline phosphatase, esterases and glycosidases, oroxidoreductases, particularly peroxidases such as horseradishperoxidase, and the like), substrates, cofactors, inhibitors,chemiluminescent groups, chromogenic agents, and calorimetric labelssuch as colloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads.

“Treat,” “treating” or “treatment” as used herein refers to any type oftreatment that imparts a benefit to a patient afflicted with a disease,including improvement in the condition of the patient (e.g., in one ormore symptoms), delay in the progression of the disease, etc.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject to achieve thetreatments described herein, without unduly deleterious side effects inlight of the severity of the disease and necessity of the treatment.

“Concurrently administering” or “concurrently administer” as used hereinmeans that the two or more compounds or compositions are administeredclosely enough in time to produce a combined effect (that is,concurrently may be simultaneously, or it may be two or more eventsoccurring within a short time period before or after each other, e.g.,sequentially). Simultaneous concurrent administration may be carried outby mixing the compounds prior to administration, or by administering thecompounds at the same point in time but at different anatomic sitesand/or by using different routes of administration.

“Internalizing factor” as used herein may be any compound or constructthat binds to a cell surface protein which is then taken up into thecell by binding. Numerous such internalizing factors are known,including but not limited to those described in D. Curiel et al., U.S.Pat. Nos. 6,274,322 and 6,022,735, the disclosures of which areincorporated herein by reference.

The definitions and techniques described herein also apply to the IL-13targeting peptides, toxin proteins, and other compounds and compositionsmentioned hereinabove and hereinbelow.

B. TARGETING PEPTIDES THAT BIND TO THE IL-13 BINDING SITE

In some embodiments of the invention, the internalizing factor,targeting protein, peptide or agent is IL-13 or a fragment thereof thatspecifically binds the IL-13 receptor. The terms “peptide,”“polypeptide,” and “protein” are used interchangeably and refer to anypolymer of amino acids (dipeptide or greater) linked through peptidebonds. Recombinant IL-13 is commercially available from a number ofsources (e.g., R&D Systems, Minneapolis, Minn., and SanofiBio-Industries, Inc., Tervose, Pa.). Alternatively, a gene or cDNAencoding IL-13 may be cloned into a plasmid or other expression vectorand expressed in any of a number of expression systems according tomethods well known to those of skill in the art. Methods of cloning andexpressing IL-13 and the nucleic acid sequence for IL-13 are well known(see, for example, Minty et al. (1993) supra and McKenzie (1987) supra).In addition, the expression of IL-13 as a component of a chimericmolecule is detailed below. Also contemplated is the use of specificIL-13 mutants or a fragment thereof as described in U.S. Pat. No.6,884,603 (Debinski et al.). An exemplary IL-13 mutant is IL-13.E13K,which has an amino acid residue at position 13 substituted for lysine.

One of skill in the art will appreciate that analogues or fragments ofIL-13 or IL-13 mutants will also specifically bind to the IL-13receptor. For example, conservative substitutions of residues (e.g., aserine for an alanine or an aspartic acid for a glutamic acid)comprising native IL-13 will provide IL-13 analogues that alsospecifically bind to the IL-13 receptor. Thus, the terms “IL-13” or“IL-13 mutant” when used in reference to a targeting molecule, alsoincludes fragments, analogues or peptide mimetics of IL-13 or IL-13mutants that also specifically bind to the IL-13 receptor. Furtherdiscussion of IL-13 as contemplated by the present invention can befound in U.S. Pat. Nos. 5,328,984 (Pastan et al.), 5,614,191 (Puri etal.), 5,919,456 (Puri et al.), 6,296,843 (Debinski), 6,428,788 (Debinskiet al.), 6,518,061 (Puri et al.), 6,576,232 (Debinski et al.), 6,630,576(Debinski), and 6,884,603 (Debinski et al.).

In some embodiments of the present invention the targeting proteinspecifically binds to the IL-13Rα2 receptor. As described above thetargeting protein that specifically binds to the IL-13Rα2 receptor maybe IL-13, a mutant of IL-13, or a fragment thereof.

The targeting peptides of the present invention can be coupled to orconjugated to effector molecules, cytosol localization elements, orsubcellular compartment localization signal elements by any suitabletechnique, including those described further in “Conjugates” below. Thedescribed conjugates can be used for therapeutic and/or diagnosticpurposes.

C. TARGETING PEPTIDES THAT DO NOT BIND TO THE IL-13 BINDING SITE

In some embodiments, the internalizing factor or targeting peptides ofthe present invention are not IL-13 or IL-13 mutants and/or fragments,but instead are peptides that do not bind to the IL-13 binding site, butinstead bind to a different binding site on the IL-13 receptor.

The single letter code for amino acids as used herein is: A (Ala), C(Cys), D (Asp), E (Glu), F (Phe), G (Gly), H (His), I (Ile), K (Lys), L(Leu), M (Met), N (Asn), P (Pro), Q (Gln), R (Arg), S (Ser), T (Thr), V(Val), W (Trp), and Y (Tyr)).

In some embodiments, targeting peptides of the present invention canhave the general formula, from amino terminus to carboxy terminus, oralternatively from carboxy terminus to amino terminus, of FORMULA I:

X—R¹—R²—R³—R⁴—R⁵—R⁵—R⁶R—⁷—Y  (I)

wherein:

R¹ is G or S;

R² is a negatively charged amino acid (for example E or D);

R³ is a large hydrophobic amino acid (for example M, W, Y, or, I);

R⁴ is a small amino acid (for example G, S or A);

R⁵ is a large or aromatic amino acid (for example W, F, H or Y);

R⁶ is a preferably hydrophobic or neutral amino acid (for example V, P,T or N);

R⁷ is a positively charged amino acid (for example R, K or H); and

X and Y are as given below.

In other embodiments, targeting peptides of the present invention canhave the general formula, from amino terminus to carboxy terminus, oralternatively from carboxy terminus to amino terminus, of FORMULA II:

X—R¹—R²—R³—R⁴—R⁵—R⁵—R⁶—R⁷—Y  (II)

wherein:

R¹ is a hydrophobic amino acid (for example L, A, I, V, or M);

R² is a preferably hydrophobic or neutral amino acid (for example P, V,T or N);

R³ is a charged or uncharged polar amino acid (for example Q, N, D, E orH)

R⁴ is a hydrophobic amino acid (for example L, A, I, V, or M);

R⁵ is large or aromatic amino acid (for example W, F, H or Y);

R⁶ is a hydrophobic amino acid (for example L, A, I, V, or M);

R⁷ is large or aromatic amino acid (for example F, W, H or Y); and

X and Y are as described below.

In still other embodiments, targeting peptides of the present inventioncan have the general formula, from amino terminus to carboxy terminus,or alternatively from carboxy terminus to amino terminus, of FORMULAIII:

X—R¹—R²—R³—R⁴—R⁵—R⁵—R⁶—R⁷—Y  (III)

wherein:

R¹ is S or G;

R² is a preferably hydrophobic or neutral amino acid (for example, P, V,T or N);

R³ is large or aromatic amino acid (for example F, W, H or Y);

R⁴ is a hydrophobic amino acid (for example, L, A, I, V, or M);

R⁵ is large or aromatic amino acid (for example H, W, F, or Y);

R⁶ is a hydrophobic amino acid (for example L, A, I, V, or M);

R⁷ is a hydrophobic amino acid (for example L, A, I, V, or M); and

X and Y are as described below.

In Formulas I-III, X and Y can each independently be present or absentand when present can each independently be a capping group, a linkinggroup (or “linker”, including non-amino acid linking groups, see, e.g.,U.S. Pat. Nos. 7,468,418; 7402,652; and 7,351,797), an amino acid (e.g.C, S or G) optionally terminated by a capping group or linking group, ora peptide consisting of from 2 to 6 or 10 additional amino acidsoptionally terminated by a capping group or linking group.

The amino acids of peptides of the invention may be in D form, L form,or a combination thereof.

Specific examples of targeting peptides of FORMULAS I-III include, butare not limited to those set forth in Tables 1-3 and Tables 4-6 below.These peptides may or may not have linking groups bonded to the carboxyterminus. Linking groups as used herein are described in more detailbelow.

Active compounds of the present invention can be produced by anysuitable means, including by synthetic organic chemical techniques or byrecombinant techniques in which a nucleic acid that encodes the activecompound is produced and introduced into a host cell (typically in theform of an expression vector) so that the encoded active compound(peptide, fusion peptide, etc.) is expressed therein. Expression vectorscan be designed for expression of proteins or polypeptides inprokaryotic or eukaryotic cells. For example, polypeptides can beexpressed in bacterial cells such as E. coli, insect cells (e.g., in thebaculovirus expression system), yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).Examples of vectors for expression in yeast S. cerevisiae includepYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).Baculovirus vectors available for expression of nucleic acids to produceproteins in cultured insect cells (e.g., Sf 9 cells) include the pAcseries (Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVLseries (Lucklow & Summers (1989) Virology 170:31-39).

Vectors can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” refer to a variety ofart-recognized techniques for introducing foreign nucleic acids (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection,electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNAcomplexes, cell sonication, gene bombardment using high velocitymicroprojectiles, and viral-mediated transfection. Suitable methods fortransforming or transfecting host cells can be found in Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory press (1989)), and other laboratory manuals.

TABLE 1 Peptides of Formula I CGEMGWVRC; (SEQ ID NO: 1) andACGEMGWVRCGGGS. (SEQ ID NO: 2)

TABLE 2 Peptides of Formula II CLPQLWLFC; (SEQ ID NO: 3) ACLPQLWLFCGGGS;(SEQ ID NO: 4)

TABLE 3 Peptides of Formula III CSPFLHLLC; (SEQ ID NO: 5) andACSPFLHLLCGGGS. (SEQ ID NO: 6)

TABLE 4 Additional Peptides of Formula I SEMGWVRC (SEQ ID NO: 7) GDMGWVR(SEQ ID NO: 8) SDWGWVR (SEQ ID NO: 9) GDYGWVR (SEQ ID NO: 10) SEIGWVRSEQ ID NO: 11) GEISWVR (SEQ ID NO: 12) GEMAWVR (SEQ ID NO: 13) GEMGFVR(SEQ ID NO: 14) GEMGHVR (SEQ ID NO: 15) GEMSYVR (SEQ ID NO: 16) GEMGWPR(SEQ ID NO: 17) GEMGWTR (SEQ ID NO: 18) GEMGWNK (SEQ ID NO: 19) GEMGWNH(SEQ ID NO: 20)

TABLE 5 Additional Peptides of Formula II APQLWLF (SEQ ID NO: 21)IPQLWLF (SEQ ID NO: 22) VPQLWLF (SEQ ID NO: 23) MPQLWLF (SEQ ID NO: 24)LVQLWLF (SEQ ID NO: 25) LTQLWLF (SEQ ID NO: 26) LNQLWLF (SEQ ID NO: 27)LPNLWLF (SEQ ID NO: 28) LPDLWLF (SEQ ID NO: 29) LPELWLF (SEQ ID NO: 30)LPHLWLF (SEQ ID NO: 31) LPQAFAW (SEQ ID NO: 32) LPQIFIH (SEQ ID NO: 33)LPQVHVY (SEQ ID NO: 34) LPQMYMY (SEQ ID NO: 35) MNHMYMY (SEQ ID NO: 36)VTEVHVH (SEQ ID NO: 37)

TABLE 6 Additional Peptides of Formula III GPFLHLL (SEQ ID NO: 38)SVFLHLL (SEQ ID NO: 39) STFLHLL (SEQ ID NO: 40) SNWLHLL (SEQ ID NO: 41)SPHLHLL (SEQ ID NO: 42) SPYLHLL (SEQ ID NO: 43) SPFAHLL (SEQ ID NO: 44)SPFIHLL (SEQ ID NO: 45) SPFVHLL (SEQ ID NO: 46) SPFMHLL (SEQ ID NO: 47)SPFLWLL (SEQ ID NO: 48) SPFLFAA (SEQ ID NO: 49) SPFLFII (SEQ ID NO: 50)SPFLHVV (SEQ ID NO: 51) SPFLYMM (SEQ ID NO: 52) GNYMYMM (SEQ ID NO: 53)GTHVFVI (SEQ ID NO: 54)

D. CONJUGATES

Targeting peptides as described herein may be coupled to or conjugatedto an effector molecule such as a diagnostic and/or therapeutic agent inaccordance with any of a variety of techniques, such as those employedin the production of immunoconjugates. See, e.g., U.S. Pat. No.6,949,245 to Sliwkowski.

In some embodiments, recombinant fusion chimera protein anti-cancercytotoxins are composed of a carrier/ligand and an effector (catalyst).Carrier/ligands can be proteinaceous compounds, such as growth factors,cytokines, and monoclonal antibodies. Among effectors, bacterial toxins,such as Pseudomonas exotoxin A and Diphtheria toxin, or plant toxins,such as ricin may be utilized in some embodiments. The fusion protein istargeted only to cells expressing a target receptor/adaptor for acarrier/ligand. These targets internalize in response to carrier/ligandbinding. Targets include, but are not limited to, protein receptors,antigens of various nature, adhesion molecules, gangliosides, etc. Forexample, EphA2 is over-expressed in a majority of patients with GBM andits ligand induces a receptor-mediated internalization once it binds thereceptor (Walker-Daniels et al. (2002) Mol. Cancer. Res. 1:79-87). Thelatter may be used for, e.g., recombinant bacterial toxin-containingcytotoxins to exert anti-tumor action (Debinski (2002) Molecular“Targeting of Brain Tumors with Cytotoxin,” In: Chimeric Toxins(Lorberboum-Galski & Lazarovici, eds., Harwood Academic Publishers) pp.222-246; Debinski (2002) Cancer Invest. 20:801-809; Debinski (2002)Cancer Invest. 20:801-809). Another non-limited example is the IL-13Rα2receptor whose ligand is internalized through receptor mediatedendocytosis.

Chemotherapeutic agents useful in the generation of such activecompounds include those described above. Conjugates of targeting peptideand one or more small molecule toxins, such as a calicheamicin, amaytansine (See U.S. Pat. No. 5,208,020), a trichothene, and CC 1065 arealso contemplated herein. In some embodiments, conjugates of targetingpeptide to Pseudomonas exotoxins are used (U.S. Pat. No. 5,328,984 toPastan et al.).

In some embodiments of the invention, the targeting peptide conjugatedto one or more maytansine molecules (e.g., about 1 to about 10maytansine molecules per targeting peptide molecule). Maytansine may,for example, be converted to May-SS-Me which may be reduced to May-SH3and reacted with modified targeting peptide (Chari et al. (1992) CancerRes. 52: 127-131) to generate an active compound.

Another conjugate of interest includes a targeting peptide conjugated toone or more calicheamicin molecules. The calicheamicin family ofantibiotics is capable of producing double-stranded DNA breaks atsub-picomolar concentrations. Structural analogues of calicheamicin thatmay be used include, but are not limited to, γ₁ ¹, α₂ ¹, α₃ ¹,N-acetyl-γ₁ ¹, PSAG and θ₁ ¹, (Hinman et al. (1993) Cancer Res.53:3336-3342; Lode et al. (1998) Cancer Res. 58:2925-2928). See alsoU.S. Pat. Nos. 5,714,586, 5,712,374, 5,264,586, and 5,773,001.

Enzymatically active toxins and fragments thereof which can be used aredescribed above and include diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain (from Corrybacteriumtyphimuriae), modeccin A chain, alpha-sarcin, Aleurites fordii proteins,dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates a conjugate formed betweenactive compounds and an antibody or a compound with nucleolytic activity(e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease;DNase).

A variety of radioactive isotopes or radionuclides are available for theproduction of radioconjugated compounds as described above.

In some embodiments, conjugates of a targeting agent and therapeuticagents or detectable groups may be made using a variety of bi-functionalprotein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin conjugate can beprepared as described in Vitetta et al. (1987) Science 238:1098.Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the targeting peptide. See WO94/11026. The linker may be a “cleavable linker” facilitating release ofthe cytotoxic drug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, dimethyl linker or disulfide-containinglinker (Chari et al. (1992) Cancer Res. 52:127-131) may be used.

Alternatively, a fusion protein including the targeting agent andtherapeutic agent or detectable group may be made, e.g. by recombinanttechniques or peptide synthesis.

In yet another embodiment, the targeting agent may be conjugated to a“receptor” (such as streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

In some embodiments, the targeting peptide is fused to a Pseudomonasexotoxin or Diphtheria toxin. (U.S. Pat. No. 5,328,984 to Pastan et al.and U.S. Pat. No. 6,296,843 to Debinski). Pseudomonas exotoxins include,but are not limited to, Pseudomonas exotoxin A (PE). The Pseudomonasexotoxin can be modified such that it substantially lacks domain Ia, andPseudomonas exotoxins may further include PE38QQR and PE4E. Diphtheriatoxins include DT390, a diphtheria toxin in which the native bindingdomain is eliminated. It will be appreciated that the toxin can beconnected to either of the amino terminus, or the carboxyl terminus.

The present invention further contemplates a fusion protein comprising,consisting of, or consisting essentially of the targeting protein and acytosol localization element, which can be made by, for example,recombinant techniques or peptide synthesis. In some embodiments thisfusion protein also comprises, consists of, or consists essentially ofan effector molecule.

“Cytosol localization element” (also referred to as an endosomal exitelement) as used herein refers to an amino acid sequence used to directa target protein, fusion protein, or fragment thereof to the cytoplasm.The amino acid sequence can be of any size and composition, for example3 to 100 amino acids in length to, 4, 5, 6, 7, 8, 10, 12, 15, 20, 25,30, 40, 50, 60, 70, 80, 90 or 100 amino acids in length. In someembodiments the cytosol localization element enables the fusion proteinor a fragment thereof to exit an endocytic compartment after beinginternalized in the process of receptor-mediated internalization andenter the cytoplasm. In some embodiments the cytosol localizationelement is proteolytically activated, such as, but not limited to, by acalcium-dependent serine endoprotease, such as furin. Exemplary cytosollocalization elements include, but are not limited to cytosollocalization elements of bacterial toxins. Such bacterial toxinsinclude, but are not limited to Pseudomonas exotoxin A (PE), Diphtheriatoxin (DT), and Ricin A chain. Additional examples are described in: B.Beaumelle et al., Selective translocation of the A chain of Diphtheriatoxin across the membrane of purified endosomes. J. Biol. Chem.267:11525-11531 (1992); I. Madshus et al., Membrane translocation ofDiphtheria toxin carrying passenger protein domain, Inf. Immun.60:3296-3302 (1992); H. Stenmark et al., Peptides fused to theamino-terminal end of Diphtheria toxin are translocated to the cytosol,J. Cell Biol. 113:1025-1032 (1991); and R. Chignola et al.,Self-potentiation of ligand-toxin conjugates containing Ricin A chainfused with viral structures, J Biol Chem 270:23345-23351 (1995). Stillother exemplary cytosol localization elements include those describe inU.S. Pat. No. 6,235,526, which is incorporated herein by reference.

The present invention further contemplates a fusion protein comprising,consisting of, or consisting essentially of the targeting protein and asubcellular compartment localization signal element, which can be madeby, for example, recombinant techniques or peptide synthesis. In someembodiments this fusion protein also comprises, consists of, or consistsessentially of a cytosol localization element and optionally an effectormolecule.

“Subcellular compartment localization signal element” as used hereinrefers to a signal sequence or tag used to direct a target protein,fusion protein, or fragment thereof to particular cellular organelles.In some embodiments the subcellular compartment localization signalelement comprises a peptide sequence. Such peptide sequences can be ofany size and composition, for example 3 to 100 amino acids in length to,4, 5, 6, 7, 8, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100amino acids in length. Exemplary cellular organelles include, but arenot limited to, the nucleus, endoplasmic reticulum, golgi apparatus,endosomes, lysosomes, peroxisomes and mitochondria. Various subcellularcompartment localization signal elements are known and/or commerciallyavailable. Exemplary subcellular compartment localization signalelements include, but are not limited to, nuclear localization signalsand lysosomal localization signals. Other exemplary subcellularcompartment localization signal elements include those described in U.S.Pat. No. 7,585,636, which is incorporated herein by reference.

“Nuclear localization signals” as used herein refers to an amino acidsequence which directs a target protein, fusion protein, or fragmentthereof into the nucleus of a cell. Generally, nuclear localizationsignals (NLS) are a class of short amino acid sequences which may beexploited for cellular import of linked or coupled cargo into thenucleus. Such amino acid sequences can be from 3 to 100 amino acids inlength to 3 to 50, 4 to 30, or 4 to 20 amino acids in length. Thenuclear localization sequences of the present invention can be: (i) amonopartite nuclear localization sequence exemplified by the SV40 largeT antigen NLS (PKKKRKV) (SEQ ID NO: 55); (ii) a bipartite motifconsisting of two basic domains separated by a variable number of spaceramino acids and exemplified by the Xenopus nucleoplasmin NLS(KRXXXXXXXXXXKKKL) (SEQ ID NO: 56); or (iii) noncanonical sequences suchas M9 of the hnRNP A1 protein, the influenza virus nucleoprotein NLS,and the yeast Gal4 protein NLS (Dingwall and Laskey, Trends Biochem Sci16:478-481, 1991). In some embodiments, the nuclear localization signalis a highly cationic or basic peptide. In other embodiments the NLScomprises two or more Arg or Lys amino acid residues. In furtherembodiments of the present invention the NLS sequence binds to cytosolicproteins, such as importins and karyopherins, which recognize andtransport NLS-containing proteins or peptides to the nuclear porecomplex. The present invention envisions the use of any nuclearlocalization signal peptide, including but not limited to, SV40 virusT-antigen NLS and NLS sequences domain derived from viral Tat proteins,such as HIV Tat. Other exemplary nuclear localization signals include,but are not limited to those discussed in Cokol et al., 2000, EMBOReports, 1(5):411-415, Boulikas, T., 1993, Crit. Rev. Eukaryot. GeneExpr., 3:193-227, Collas, P. et al., 1996, Transgenic Research, 5:451-458, Collas and Alestrom, 1997, Biochem. Cell Biol. 75: 633-640,Collas and Alestrom, 1998, Transgenic Research, 7: 303-309, Collas andAlestrom, 1996, Mol. Reprod. Devel., 45:431-438, and U.S. Pat. Nos.7,531,624, 7,498,177, 7,332,586, and 7,550,650, all of which areincorporated by reference in their entireties.

“Lysosomal localization signal” as used herein refers to an amino acidsequence which directs a target protein or fusion protein to lysozymes.Examples include, but are not limited to, lysosome associated membraneprotein 1 (LAMP-1) tail sequence: RKRSHAGYQTI (SEQ ID NO: 57); lysosomalacid phosphatase (LAP): RLKRMQAQPPGYRHVADGEDHAV (SEQ ID NO: 58), andlysosomal integral membrane protein 2 (LIMP-2): RGQGSTDEGTADERAPLIRT(SEQ ID NO: 59).

In some embodiments of the present invention the fusion proteincomprises, consists of, or consists essentially of a targeting protein,a cytosol localization element, a subcellular compartment localizationsignal element, and optionally an effector molecule. These componentsmay be coupled to one another in any order that allows for the targetingprotein to bind to its receptor and further allows for the transport ofthe fusion protein or a fragment thereof into the nucleus.

In further embodiments of the present invention the fusion proteincomprises, consists of, or consists essentially of a targeting proteincomprising IL-13, a mutant of IL-13 or an analogue or fragment thereof;a cytosol localization element comprising Pseudomonas exotoxin A (PE) orDiphtheria toxin (DT); and a subcellular compartment localization signalelement comprising a nuclear localization signal or a lysosomallocalization signal. In other embodiments of the present invention thefusion protein comprises, consists of, or consists essentially ofIL-13.E13K, the cytosol bacterial toxin domain D2 of PE, and a nuclearlocalization signal from the SV40 T antigen. In one embodiment of thepresent invention the fusion protein is a single-chain recombinantprotein comprising, consisting of, or consisting essentially of, fromthe N-terminus to the C-terminus, IL-13.E13K, the cytosol bacterialtoxin domain D2 of PE, and a nuclear localization signal from the SV40 Tantigen, i.e. IL-13.E13K-D2-NLS.

E. PHARMACEUTICAL FORMULATIONS AND METHODS

The active compounds, conjugates, and/or compositions thereof describedherein may be formulated for administration in a pharmaceutical carrierin accordance with known techniques. See, e.g., Remington, The Scienceand Practice of Pharmacy (9^(th) Ed. 1995). In the manufacture of apharmaceutical formulation according to the invention, the activecompound(s) (including the physiologically acceptable salts thereof) istypically admixed with, inter alia, an acceptable carrier. The carriermust, of course, be acceptable in the sense of being compatible with anyother ingredients in the formulation and must not be deleterious to thepatient. The carrier may be a solid or a liquid, or both, and ispreferably formulated with the compound(s) as a unit-dose formulation,for example, a tablet, which may contain from 0.01 or 0.5% to 95% or 99%by weight of the active compound. One or more active compounds may beincorporated in the formulations of the invention, which may be preparedby any of the well-known techniques of pharmacy comprising admixing thecomponents, optionally including one or more accessory ingredients.

The formulations of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g.,subcutaneous, intramuscular, intradermal, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

Particular routes of parenteral administration include intrathecalinjection, including directly into the tumor or a tumor resectioncavity, and intraventricular injection into a ventricle of the brain.

Active compounds and compositions may be administered by intratumorinjection (including tumors in any region such as tumors of the brain),or in the case of brain tumors injection into a ventricle of the brain.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes thatrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising an active compoundor composition in a unit dosage form in a sealed container. The compoundor composition is provided in the form of a lyophilizate that is capableof being reconstituted with a suitable pharmaceutically acceptablecarrier to form a liquid composition suitable for injection thereof intoa subject. The unit dosage form typically comprises from about 10 mg toabout 10 grams of the compound or composition. When the compound orcomposition is substantially water-insoluble, a sufficient amount ofemulsifying agent that is physiologically acceptable may be employed insufficient quantity to emulsify the compound or composition in anaqueous carrier. One such useful emulsifying agent is phosphatidylcholine.

Further, the present invention provides liposomal formulations of thecompounds disclosed herein and compositions thereof. The technology forforming liposomal suspensions is well known in the art. When thecompound or composition thereof is an aqueous-soluble composition, usingconventional liposome technology, the same may be incorporated intolipid vesicles. In such an instance, due to the water solubility of thecompound or composition, the compound or composition will besubstantially entrained within the hydrophilic center or core of theliposomes. The lipid layer employed may be of any conventionalcomposition and may either contain cholesterol or may becholesterol-free. When the compound or composition of interest iswater-insoluble, again employing conventional liposome formationtechnology, the composition may be substantially entrained within thehydrophobic lipid bilayer that forms the structure of the liposome. Ineither instance, the liposomes that are produced may be reduced in size,as through the use of standard sonication and homogenization techniques.

Liposomal formulations containing the compounds disclosed herein orcompositions thereof (e.g., IL-13 conjugates, such asIL-13.E13K-D2-NLS), may be lyophilized to produce a lyophilizate, whichmay be reconstituted with a pharmaceutically acceptable carrier, such aswater, to regenerate a liposomal suspension. Examples of liposomalformulations that can be used include the neutral lipid1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DPOC) (See, e.g., LandenJr. et al. (2005) Cancer Res. 65:6910-6918).

Other pharmaceutical compositions may be prepared from thewater-insoluble compounds disclosed herein, or compositions thereof,such as aqueous base emulsions. In such an instance, the compositionwill contain a sufficient amount of pharmaceutically acceptableemulsifying agent to emulsify the desired amount of the compound orcomposition thereof. Particularly useful emulsifying agents includephosphatidyl cholines, and lecithin.

In addition to active compounds, the pharmaceutical compositions maycontain other additives, such as pH-adjusting additives. In particular,useful pH-adjusting agents include acids, such as hydrochloric acid,bases or buffers, such as sodium lactate, sodium acetate, sodiumphosphate, sodium citrate, sodium borate, or sodium gluconate. Further,the compositions may contain microbial preservatives. Useful microbialpreservatives include methylparaben, propylparaben, and benzyl alcohol.The microbial preservative is typically employed when the formulation isplaced in a vial designed for multidose use. Of course, as indicated,the pharmaceutical compositions of the present invention may belyophilized using techniques well-known in the art.

The therapeutically effective dosage of any one active agent, the use ofwhich is in the scope of present invention, will vary somewhat fromcompound to compound, and patient to patient, and will depend uponfactors such as the age and condition of the patient and the route ofdelivery. Such dosages can be determined in accordance with routinepharmacological procedures known to those skilled in the art.

As a general proposition, the initial pharmaceutically effective amountof the active compound or composition administered parenterally will bein the range of about 0.1 to 50 mg/kg of patient body weight per day,with the typical initial range of antibody used being 0.3 to mg/kg/day,more preferably 0.3 to 15 mg/kg/day. The desired dosage can be deliveredby a single bolus administration, by multiple bolus administrations, orby continuous infusion administration of active compound, depending onthe pattern of pharmacokinetic decay that the practitioner wishes toachieve.

The active compound(s) is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 kg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of activecompound(s) is an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. A typical daily dosage might range from about0.1, 0.5, 1, 10 or 100 kg/kg up to 100, 200 or 500 mg/kg, or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs. Amore particular dosage of the active compound will be in the range fromabout 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) maybe administered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g., such thatthe patient receives from about two to about twenty, e.g. about sixdoses of the anti-ErbB2 antibody). An initial higher loading dose,followed by one or more lower doses may be administered. An exemplarydosing regimen comprises administering an initial loading dose of about0.5 to 10 mg/kg, followed by a weekly maintenance dose of about 0.5 to10 mg/kg of the active compound. However, other dosage regimens may beuseful. The progress of this therapy is easily monitored by conventionaltechniques and assays.

Subjects treated by the methods of the present invention can also beadministered one or more additional therapeutic agents. See U.S. Pat.No. 5,677,178. Chemotherapeutic agents may be administered by methodswell known to the skilled practitioner, including systemically, directinjection into the cancer, or by localization at the site of the cancerby associating the desired chemotherapeutic agent with an appropriateslow release material or intra-arterial perfusing of the tumor. Thepreferred dose may be chosen by the practitioner based on the nature ofthe cancer to be treated, and other factors routinely considered inadministering. See, e.g., U.S. Pat. No. 7,078,030.

Subjects may also be treated by radiation therapy, including, but notlimited to, external beam radiotherapy, which may be at any suitabledose (e.g., 20 to 70 Gy or more per tumor, typically delivered over afractionated schedule).

Pharmaceutical compositions containing targeting agent in unlabeled formmay be administered to subjects as blocking reagents, in like manner asdescribed in Abrams et al., U.S. Pat. No. RE38,008, in conjunction withthe administration of targeting agent coupled to a therapeutic group.

Targeting peptide coupled to a diagnostic group may also be used invitro as histological reagents on tissue samples, where binding of theIL-13 receptor is indicative of cancer tissue in the tissue sample.

Examples Materials and Methods

Cell culture. Human GBM cell lines U-251 MG and LN229 were obtained fromAmerican Type Culture Collection (Manassas, Va.). U-251 MG cells weremaintained in DMEM (Lonza, Walkersville, Md.) supplemented with 1×non-essential amino acid (Invitrogen, Carlsbad, Calif.) and 10% FCS(Hyclone, Logan, Utah). LN229 cells were grown in DMEM supplemented with10% FCS. G48a cells were grown and maintained in RPMI 1640 (Lonza,Walkersville, Md.) supplemented with glucose, adjusted to 4 gm/litre ofmedia and 10% FCS (13).

Cloning, production and purification of targeted proteins. A duplexprimer cloning strategy was employed wherein SV40 T-antigen NLS 5′ and3′ sequence primers were synthesised (Invitrogen) and made into duplexDNA (containing XhoI/BamHI ends) by incubating the primers in favorableannealing conditions. The annealed duplex was then subcloned into theIL-13-D2 containing plasmid using XhoI/BamHI at the 3′ end to produceIL-13-D2-NLS. The IL-13-D2 plasmid was engineered by sub-cloning it froma previously generated IL-13-D2-PE38QQR plasmid (24). The IL-13 mutantrecombinant constructs were made by replacing the wild type IL-13sequence from the parent plasmid with the mutant IL-13 sequence (25).The NH2-terminal end of NLS domain was joined to the COOH terminal ofIL-13.E13K domain using the HindIII site to form the IL-13.E13K-NLSplasmid. Also, all of these recombinant constructs were transformed inDH5a E. coli cells for amplification. All the constructs were sequencedat DNA sequencing Laboratory of the Comprehensive Center of Wake ForestUniversity and analyzed for their in-frame DNA sequence using anautomated sequence analyzer prior to protein expression.

Also, the IL-13/IL-13.E13K-D2-NLS and other control DNA constructs havebeen created in a manner such that it enables the expression of theseproteins under the IPTG inducible T7 promoter in BL21 (λDE3) E. coliprotein expression system as previously described (33). In brief, therecombinant constructs were transformed in BL21 cells and the cells weregrown in Luria-broth media supplemented with 100 μg/ml of ampicillin at37° C. shaker. When the A600 of the bacterial culture media reachedaround 1.4, the recombinant protein expression in the cells was inducedby addition of 1 nmol/L of IPTG and allowed to incubate for further 90min. The expressed proteins in the inclusion bodies were then denaturedusing 7 M Guanidine (MP Biomedicals, Salon, Ohio) and1,4-Dithiothr.eitol (Sigma, St. Louis, Mo.). The reduced protein wasthen renatured in a buffer containing arginine/L-glutathione oxidase(Sigma, St. Louis, Mo.). The protein was further dialyzed and purifiedby SP Sepharose ion-exchange liquid chromatography (GE Healthcare,Piscataway, N.J.) using Fast Protein Liquid Chromatography system (GEHealthcare, Piscataway, N.J.). The purified proteins were subsequentlyrun on SDS-PAGE gels to identify the purity of the isolated proteins.All the proteins obtained were >90% pure.

Colorimetric MTS/PMS cell viability assay. 1×103 U-251 MG cells wereplated per well in quadruplicates for each concentration to be tested.IL-13.E13K-PE38QQR is an IL-13Rα2 based cytotoxin against GBM (24).After 24 hours incubation at 370 C. for the cells to attach, a fixedconcentration (i.e. 1 μM) of the IL13.E13K-D2-NLS and other purifiedproteins were added and incubated at 37° C. for 1 hr. After 1 hr.incubation, increasing concentrations of the IL-13.E13K-PE38QQR rangingfrom 0.1 to 100 ng/ml was added and the plate was incubated for 48 hr.Cells treated with cyclohexamide and just the cytotoxins were used ascontrols. After 48 hr., cell viability was measured using the MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]/PMS[phenazine methosulfate] dye (Promega, Madison, Wis.) as per themanufacturer's instructions. The absorbance from the assay was measuredat 490 nm using the plate reader Spectra max 340 PC (Molecular Devices,Sunnyvale, Calif.) and data was plotted as percentage of control versusconcentration of the toxin used.

IL-13-D2-NLS and IL-13-D2 labeling with EDC-Sulpho-NHS and Alexa fluor488 labels. Purified IL-13-D2-NLS and the IL-13-D2 proteins were labeledat their carboxylate amino acids via EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). EDCreacts with a carboxyl group on an amino acid of the protein and formsan amine reactive O-acylisourea intermediate that swiftly reacts with anamino group to form an amide bond and release the isourea by-product.The intermediate is unstable in aqueous solutions and therefore,two-step conjugation procedures require N-hydroxysuccinimidestabilization (Sulfo-NHS).

Sulfo-NHS reacts with the O-acylisourea intermediate and stabilizes it.Next, Alexa fluor 488-hydrazide was added, which replaced the Sulfo-NHSand formed a stable amide bond on the carboxyl groups of the protein toform labeled protein conjugates.

The proteins were initially dissolved in the activation buffer (0.05 MMES, 0.5 M NaCl, pH 6) at the concentration of 1 mg/ml usingbuffer-exchange columns. Later 2 mM EDC and 5 mM Sulfo-NHS (ThermoScientific, Waltham, Mass.) were added to the proteins and allowed toreact for 15 min at RT. Subsequently, 0.14 μl of 2-mercaptoethanol wasadded to quench the unreacted EDC. The protein-EDC-Sulfo-NHS conjugateswere then dissolved in the coupling buffer (0.1 M sodium phosphate, 0.15M NaCl, pH 7.5) using buffer exchange columns (Pierce, Rockford, Ill.).Next, alexa fluor 488 hydrazide (dissolved in the coupling buffer)(Invitrogen, Carlsbad, Calif.) was added at 25 molar concentrationexcess to the proteins and incubated in the dark at RT for 30 minutes.After incubation, 10 mM hydroxylamine (Thermo Scientific, Rockford,Ill.) was added to quench the excess fluor. The excess unreactedhydrazide fluor was removed using Pierce protein desalting columns.

Localization studies of the labeled proteins on IL-13Rα2 positive U-251MG cells using Alexafluor 488 EDC-Sulfo-NHS labeled proteins. 25,000U-251 MG GBM cells were plated on coverslip/per well in a 24-well plate.The cells were allowed to adhere to the coverslips for 24 hr., afterwhich 500 nM each of the EDC-Sulpho-NHS labeled proteins were added tothe U-251 MG cells for 15 min and 4 hr. After the incubations, the cellswere fixed with acetone (pre-chilled at −20° F.) for 10 mins and washedwith PBS 4× times. The coverslips were then mounted on the slides usingthe gel mount (Biomeda, Foster City, Calif.) and observed with LSM 510Zeiss Confocal Microscope (Cellular Imaging Core, Comprehensive CancerCenter, Wake Forest University) and the images processed using Zeiss LSMImage Browser (version 4.2).

Direct labeling of IL-13.E13K-D2-NLS and IL-13.E13K-D2 with Alexa fluorlabels. The proteins were directly labeled with alexa fluor 488 dyeusing the Alexa fluor 488 microscale protein labeling kit fromInvitrogen (Carlsbad, Calif.) as per the manufacturer's instructions. Amolar ratio of 25 of the dye to the protein was used to label both theproteins. The proteins were run on 12% SDS-PAGE gel. The gel was scannedusing Typhoon 9210 (Amersham Pharmacia Biotech) for fluorescence signalsand later stained using coomassie blue dye.

Localization studies of the labeled proteins on IL-13Rα2 positive U-251MG cells for Alexa fluor 488 directly labeled proteins. 25,000 U-251 MGGBM cells were plated on coverslips per well in a 24-well plate. After24 hr. for allowing the cells to adhere and attach to the plate, 1μM/well of alexa fluor directly labeled proteins were added to the U-251MG cells for 15 min and 4 hr. After the incubations, the cells werefixed with 5% paraformaldehyde (Ted Pella, Redding, Calif.) for 15 minsat 37° C. and washed with 1×PBS (3 times). The cells were thenpermeabilized with 0.1% Triton-X-100/0.2% BSA-PBS for 10 min at RT.After permeabilization, the cells were washed 3 times with 1×PBS.Subsequently, Topro-3 iodide (Invitrogen, Carlsbad, Calif.) was added ata concentration of 1:1000 dilution of the 1 mM stock to stain the cellnuclei. The coverslips were then mounted on the slides using the gelmount (Biomeda, Foster City, Calif.) and observed with LSM 510 ZeissConfocal Microscope (Cellular Imaging Core, Comprehensive Cancer Center,Wake Forest University) and the images processed using Zeiss LSM ImageBrowser (version 4.2).

Direct labeling of IL-13.E13K-D2-NLS and IL-13.E13K-D2 with biotin andtyramide signal amplification system. Biotin-XX microscale proteinlabeling kit (Invitrogen, Carlsbad, Calif.) was used to label theproteins as per the manufacturer's instructions. A different molar ratioof 12, 8 or 4 biotin-dye to the proteins was used. The biotin-labeledproteins were run on a gel and a western blot carried out usingstreptavidin-HR.P (Pierce, Rockford, Ill.) to detect for biotin-labeledproteins. The number of Biotin molecules attached to the proteins wasdetermined by the Biofluoreporter assay kit (Invitrogen) as per themanufacturer's guidelines.

Localization studies of the labeled proteins on IL-13Rα2 positive U-251MG cells for biotin-conjugated proteins. 12,500 U-251 MG GBM cells wereplated on coverslips per well in a 24-well plate. After 24 hr., 1μM/well of biotin-labeled proteins was added onto the U-251 MG cells for15 min, 4, 8 and 24 hr. After the incubations, the cells were fixed with4% paraformaldehyde (Ted Pella, Redding, Calif.) for 15 mins at 37° C.and washed with PBS 4× times. The cells were then permeabilized with0.1% Triton-X-100/0.2% BSA-PBS for 10 min at RT. After permeabilization,the cells were washed 3 times with 1×PBS. After washings, Tyramidesignal amplification kit (Invitrogen, Carlsbad, Calif.) using Alexafluor 488 dyes and HR.P-streptavidin was carried out as permanufacturer's instructions. Topro-3 iodide (Invitrogen, Carlsbad,Calif.) was added at a concentration of 1:1000 dilution of the 1 mMstock to stain the cell nuclei. After the tyramide staining, wells werewashed and mounted with gel mount (Biomeda, Foster City, Calif.) andobserved with LSM 510 Zeiss Confocal Microscope (Cellular Imaging Core,Comprehensive Cancer Center, Wake Forest University) and the imagesprocessed using Zeiss LSM Image Browser (version 4.2).

Immunoblotting. 500 ng/well of each of the recombinant biotin conjugatedproteins were loaded onto a 12% SDS-PAGE gel and transferred to apolyvinylidene difluoride membrane (Perkin Elmer, Shelton, Conn.). Blotswere blocked with 5% milk-phosphate buffered saline (PBS) for 1 hr. atroom temperature (RT). Biotin-proteins were detected using streptavidinconjugated with horseradish peroxidase (Thermo Fisher Scientific,Rockford, Ill.) diluted 1:16000 in blocking buffer. The detection wasperformed using an ECL plus kit (GE Healthcare).

Results

Production of IL-13.E13K-D2-NLS, IL-13.E13K-D2, IL-13.E13K-NLS andIL-13.E13K proteins. We aim at developing effective drug/radioactiveisotope delivery vehicles to specific intracellular compartments of acancer cell, based preferentially on recombinant proteins. Hence, wehave developed here a recombinant protein delivery vehicle to the nucleiof GBM cells. This delivery vehicle recognizes the IL-13Rα2, which isoverexpressed on GBM cells. The IL-13.E13K-D2-NLS recombinant proteinrecognizes IL-13Rα2 and is internalized into the GBM cells, exitsendosomes and is trafficked to the cell's nuclei. IL-13.E13K-D2-NLS andits control proteins, IL-13.E13K-NLS and IL-13.E13K-D2 as well asIL-13.E13K, which are not expected to either leave the endosomalcompartment or reach the nucleus, respectively were produced in E. coliand purified using the FPLC system. IL-13.E13K-D2-NLS was highlyinducible in BL21 E. coli cells. The induced protein was isolated andfurther processed using a disulphide-shuffling method and purified usingFPLC column, as described previously (25; 34). Even with the first stepof purification, the protein was highly purified. The controlIL-13.E13K-D2, the IL-13.E13K-NLS and the IL-13.E13K recombinantproteins were expressed, processed and purified in a similar manner.

IL-13.E13K-D2-NLS, IL-13.E13K-D2, IL-13.E13K-NLS and IL-13.E13K competefor IL-13Rα2 on GBM cells. We next wished to confirm that all thepurified recombinant proteins bind to the IL-13Rα2 receptor on GBMcells. To this end, we carried out a cell-viability assay in which theserecombinant proteins bound to the IL-13Rα2 receptor and protectedagainst cytotoxic action of IL-13.E13K-PE38QQR. IL-13.E13K-PE38QQR, asmentioned earlier, is a recombinant cytotoxin that binds to theIL-13Rα2, is internalized into cells leading to cell killing through thecleaved active portion of PE, enzymatic domain III. As expected, allrecombinant proteins of interest blocked the action of the cytotoxin,resulting in no cell killing: IL13.E13K-D2-NLS; IL-13.E13K-D2;IL-13.E13K-NLS and IL-13.E13K. These results confirm that all therecombinant proteins retain IL-13.E13K ligand binding properties andcompete specifically for the IL-13Rα2.

IL-13.E13K-D2-NLS localizes to the nuclei of U-251 MG GBM cells. Next,we wished to monitor the intracellular journey as well as thesubcellular localization of our targeted proteins. To this end, wefluorescently labeled these proteins using three differentapproaches/methods. For the first approach, we labeled the carboxylamino acids of the proteins, so as not to modify the primary amines(lysines) present in the NLS domain of the protein. Thus, we utilizedthe Sulfo-NHS-EDC and Alexa fluor 488 labeling techniques. IL-13-D2-NLSand IL-13-D2 were labeled at their carboxylate groups on amino acidswith alexa fluor 488 via EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride andSulfo-NHS (N-hydroxysuccinimide) (see Materials and Methods). In thesecond approach, we directly labeled the primary amines of the proteins,which are present also in the lysines, with the Alexa Fluor 488-TFPreactive dyes. And for the third approach, we carried out an indirectlabeling method; here we initially conjugated our proteins at theprimary amines with biotin molecules to make biotinylated-proteins.These biotinylated-proteins were then used for cell localizationexperiments, later the biotin molecules were detected usingHRP-Streptavidin and the signal amplified using Tyramide signalamplification method.

After labeling the proteins using the first conjugation method, i.e.EDC-Sulfo-NHS Alexa fluor 488 labeling, we performed cell localizationexperiments in U-251 MG GBM cells. We observed that the IL-13-D2-NLSeffectively localized to the nucleus at 1 hr., preceded by membrane andcytosolic localization at 15 min. We also performed Z-stack analysis toconfirm the localization of the protein inside the nucleus. The Z-stackanalysis demonstrated a nuclear localization of IL-13.E13K-D2-NLS (datanot shown). On the other hand, IL-13-D2 did get internalized into theU-251 MG cells and was found to be in the cytosol, primarily in theperi-nuclear region, but it did not travel into the nucleus at either 15min or 1 hr. of the experiment.

The above experiments strongly suggested an ability of IL-13-D2-NLS, butnot IL-13-D2, to localize to U-251 GBM cells' nuclei. However, in orderto obtain higher resolution pictures, we carried out a direct labelingof IL-13.E13K-D2-NLS and IL-13.E13K-D2 with the Alexa-fluor 488 dye. Inthis second approach, Alexa fluor 488 tetrafluorophenyl (TFP) reactivedye molecules attach directly to the primary amines of the amino acidsof the proteins forming stable protein-dye conjugates. The protein-dyeconjugates were visualized using either Coomassie-stained SDS-PAGE orfluorescence signals using Typhoon imaging system. The Typhoon scanshowed protein-dye conjugates emitting fluorescence signals, while theunconjugated proteins did not produce any signals (not shown). We nextrepeated the localization experiments in U-251 MG cells and found whatwe had observed earlier. IL-13.E13K-D2-NLS localized to the nuclei at 1hr. (not shown) whereas IL-13.E13K-D2 protein never trafficked into thenucleus.

In order to examine whether yet another visualization method woulddocument the same nuclei-localization ability of our recombinantconstructs; we decided to use a signal amplification method via tyramidemolecules. We initially labeled our proteins using biotin-XXsulfosuccinimidyl ester (biotin-XX, SSE); which reacts very efficientlywith the primary amines of the proteins forming stable protein-biotinconjugates. The biotinylated proteins were analyzed usingSDS-PAGE/Western blot and the protein-biotin conjugates were detectedusing streptavidin-HRP. The Western blot indicated that both theproteins had been biotinylated. The number of biotins on each of theseproteins was quantified by performing FluoReporter Biotin Quantitationassay based on standard curve. Using a quadratic fit equation, theIL-13.E13K-D2-NLS and IL-13.E13K-D2 had a similar degree of labeling(DOL) of 13.87 and 14.45 when labeled at a protein to dye molar ratio of1:4 and 1:8, respectively. Next, these biotinylated proteins were testedin a neutralization of cytotoxicity assay (not shown). BothIL-13.E13K-D2-NLS and IL-13.E13K-D2 biotinylated proteins blocked theaction of IL-13Rα2-specific cytotoxin-mediated U-251 MG cell killingindicating that these conjugates still compete for the receptor afterundergoing biotinylation. The cell localization experiment was thenconducted and the proteins were detected using Alexa fluor 488 andHRP-Streptavidin tyramide signal amplification procedure (see theMethods section). We found that in the case of IL-13.E13K-D2-NLS, at 5min., the protein was bound to the cell membrane with some cytosoliclocalization. At 4 hr., cells had nuclear localization, whereas almostall the cells had a significant portion of the protein inside theirnuclei at 8 hr. and 24 hr. For the IL-13.E13K-D2, at 5 min the proteinwas mostly found bound to the cell membrane with some moleculesundergoing internalization. Whereas, at 8 and 24 hr. the protein waspredominantly internalized and localized in the perinuclear region ofcells. At 4 hr., the IL-13.E13K-D2 protein had cytosolic localization.Z-stack analyses of a 24 hr. experiment (not shown) establishes that theIL-13.E13K-D2 protein does not migrate to the nucleus.

We have also carried out experiments wherein we have labeled theseproteins with different molar ratios of the biotin-dye. The protein:dyeratios used were 12, 8 and 4. When both IL13.E13K-D2-NLS andIL-13.E13K-D2 proteins were labeled at dye molar ratio of 12, weobserved similar localization for these proteins as described, except wedid not observe any nuclear localization at 4 hours. When we went downon the amount of dye (protein:dye molar ratio of 8 and 4 respectively)we observed more cells having nuclear localization at 4 hr. (Data notshown).

We also carried out cell localization experiment with another controlprotein, IL-13.E13K-NLS, which is devoid of Domain 2 of PE and shouldnot be able to undergo endosomal translocation and subsequent nucleartransport; it should behave like the IL-13.E13K ligand alone. 12% SDSPAGE/Western blot of the IL-13.E1K-D2-NLS, IL-13.E13K-D2, IL-13.E13K-NLSand the IL-13.E13K proteins conjugated with biotin and probed withstreptavidin-HRP indicate that all the proteins are similarly labeledwith biotin and also all the biotin-conjugated proteins bind to theIL-13Rα2 on GBM cells. The studies with IL-13.E13K-NLS and IL-13.E13Kindicated our hypothesis to be correct, since this control protein diddemonstrate perinuclear localization, but no nuclear transport at 24 hr.The same was observed in the cell localization studies using just theIL-13.E13K ligand. The IL-13.E13K accumulated mainly in the peri-nuclearregion. Very few cells had these proteins in the nucleus. We have alsocarried out the localization studies for the IL-13.E13K-NLS andIL-13.E13K at 8, 4 hr and 5 min and they all demonstrate the resultsobserved at 24 hr. (Data not shown).

IL-13.E13K-D2-NLS localizes to the nucleus of G48a GBM cells. Werepeated the above experiments in another GBM cell line, G48a (13),which over-expresses IL-13Rα2. We obtained similar results as with theU-251 MG cells. Again, almost all the cells had the IL-13.E13K-D2-NLSprotein inside their nuclei not only at 8 hr. and 24 hr., but already at4 hr. of the experiment. Again, at 5 min, we observed mainly plasmamembrane binding with some internalization of the protein. Z-stackanalysis for the 24 hr. experiment establishes nuclear localization ofthe IL-13.E13K-D2-NLS protein. Similar results were observed for theIL-13.E13K-D2, IL-13.E13K-NLS and IL-13.E13K proteins in G48 cells as inU-251 MG cells; IL-13.E13K-D2, as well as IL-13.E13K-NLS and IL-13.E13Kwere not found to have any nuclear localization at any of thetime-points and mainly had cytosolic/perinuclear localization with thetime of experiment at 4, 8 and 24 hr. and cell membrane binding at 5min.

IL-13.E13K-D2-NLS does not localizes to the nucleus of LN229 cells. Wecarried out identical experiments with biotin-labeled IL-13.E13K-D2-NLSin LN229 cells, very low expressors of IL-13Rα2. We observed that theprotein displayed some binding to the cell surface with moderateinternalization, but we did not observe any nuclear localization for theIL-13.E13K-D2-NLS protein at any of the experimental time points and nocytosolic or perinuclear localization for the IL-13.E13K-D2,IL-13.E13K-NLS and IL-13.E13K at 24 hr. contrary to what we observed inIL-13Rα2 high expressors, U-251 MG and G48a cells. Z-stack analysis forthe IL-13.E13K-D2-NLS protein localization in an LN229 cell at 24 hr.depicts low internalization and no nuclear localization for the proteinin these cells.

REFERENCES

-   1. Debinski W, Siegall C B, FitzGerald D, Pastan I. Substitution of    foreign protein sequences into a chimeric toxin composed of    transforming growth factor alpha and Pseudomonas exotoxin. Mol. Cell    Biol. 1991 March; 11(3):1751-3.-   2. Debinski W, Pastan I. Monovalent immunotoxin containing truncated    form of Pseudomonas exotoxin as potent antitumor agent. Cancer Res.    1992 Oct. 1; 52(19):5379-85.-   3. Debinski W, Obiri N I, Pastan I, Puri R K. A novel chimeric    protein composed of interleukin 13 and Pseudomonas exotoxin is    highly cytotoxic to human carcinoma cells expressing receptors for    interleukin 13 and interleukin 4. J. Biol. Chem. 1995 Jul. 14;    270(28):16775-80.-   4. Chiron M F, Fryling C M, FitzGerald D. Furin-mediated cleavage of    Pseudomonas exotoxin-derived chimeric toxins. J. Biol. Chem. 1997    Dec. 12; 272(50):31707-11.-   5. Inocencio N M, Moehring J M, Moehring T J. Furin activates    Pseudomonas exotoxin A by specific cleavage in vivo and in vitro. J.    Biol. Chem. 1994 Dec. 16; 269(50):31831-5.-   6. Moehring J M, Inocencio N M, Robertson B J, Moehring T J.    Expression of mouse furin in a Chinese hamster cell resistant to    Pseudomonas exotoxin A and viruses complements the genetic    lesion. J. Biol. Chem. 1993 Feb. 5; 268(4):2590-4.-   7. Ogata M, Chaudhary V K, Pastan I, FitzGerald DJ. Processing of    Pseudomonas exotoxin by a cellular protease results in the    generation of a 37,000-Da toxin fragment that is translocated to the    cytosol. J. Biol. Chem. 1990 Nov. 25; 265(33):20678-85.-   8. Jinno Y, Ogata M, Chaudhary V K, Willingham M C, Adhya S,    FitzGerald D, Pastan I. Domain II mutants of Pseudomonas exotoxin    deficient in translocation. J. Biol. Chem. 1989 Sep. 25;    264(27):15953-9.-   9. Siegall C B, Ogata M, Pastan I, FitzGerald DJ. Analysis of    sequences in domain II of Pseudomonas exotoxin A which mediate    translocation. Biochemistry 1991 Jul. 23; 30(29):7154-9.-   10. London S D, Schmaljohn A L, Dalrymple J M, Rice C M. Infectious    enveloped RNA virus antigenic chimeras. Proc. Natl. Acad. Sci. U.S.A    1992 Jan. 1; 89(1):207-11.-   11. Stenmark H, Moskaug J O, Madshus I H, Sandvig K, Olsnes S.    Peptides fused to the amino-terminal end of diphtheria toxin are    translocated to the cytosol. J. Cell Biol. 1991 June;    113(5):1025-32.-   12. Stupp R, Dietrich P Y, Ostermann K S, Pica A, Maillard I, Maeder    P, Meuli R, Janzer R, Pizzolato G, Miralbell R, et al. Promising    survival for patients with newly diagnosed glioblastoma multiforme    treated with concomitant radiation plus temozolomide followed by    adjuvant temozolomide. J. Clin. Oncol. 2002 Mar. 1; 20(5):1375-82.-   13. Debinski W, Gibo D M. Fos-related antigen 1 modulates malignant    features of glioma cells. Mol. Cancer Res. 2005 April; 3(4):237-49.-   14. Mintz A, Gibo D M, Slagle-Webb B, Christensen N D, Debinski W.    IL-13Ralpha2 is a glioma-restricted receptor for interleukin-13.    Neoplasia. 2002 September; 4(5):388-99.-   15. Mintz A, Gibo D M, Madhankumar A B, Cladel N M, Christensen N D,    Debinski W. Protein- and DNA-based active immunotherapy targeting    interleukin-13 receptor alpha2. Cancer Biother. Radiopharm. 2008    October; 23(5):581-9.-   16. Wykosky J, Gibo D M, Stanton C, Debinski W. EphA2 as a novel    molecular marker and target in glioblastoma multiforme. Mol. Cancer    Res. 2005 October; 3(10):541-51.-   17. Wykosky J, Gibo D M, Stanton C, Debinski W. Interleukin-13    receptor alpha 2, EphA2, and Fos-related antigen 1 as molecular    denominators of high-grade astrocytomas and specific targets for    combinatorial therapy. Clin. Cancer Res. 2008 Jan. 1; 14(1):199-208.-   18. Debinski W. Drug cocktails for effective treatment of    glioblastoma multiforme. Expert. Rev. Neurother. 2008 April;    8(4):515-7.-   19. Debinski W, Gibo D M, Hulet S W, Connor J R, Gillespie G Y.    Receptor for interleukin 13 is a marker and therapeutic target for    human high-grade gliomas. Clin. Cancer Res. 1999 May; 5(5):985-90.-   20. Saikali S, Avril T, Collet B, Hamlat A, Bansard J Y, Drenou B,    Guegan Y, Quillien V. Expression of nine tumour antigens in a series    of human glioblastoma multiforme: interest of EGFRvIII,    IL-13Ralpha2, gp100 and TRP-2 for immunotherapy. J. Neurooncol. 2007    January; 81(2):139-48.-   21. Debinski W, Gibo D M. Molecular expression analysis of    restrictive receptor for interleukin 13, a brain tumor-associated    cancer/testis antigen. Mol. Med. 2000 May; 6(5):440-9.-   22. Bernard J, Treton D, Vermot-Desroches C, Boden C, Horellou P,    Angevin E, Galanaud P, Wijdenes J, Richard Y. Expression of    interleukin 13 receptor in glioma and renal cell carcinoma:    IL13Ralpha2 as a decoy receptor for IL13 1. Lab Invest 2001    September; 81(9):1223-31.-   23. Kawakami K, Taguchi J, Murata T, Puri R K. The interleukin-13    receptor alpha2 chain: an essential component for binding and    internalization but not for interleukin-13-induced signal    transduction through the STAT6 pathway. Blood 2001 May 1;    97(9):2673-9.-   24. Debinski W, Obiri N I, Powers S K, Pastan I, Puri R K. Human    glioma cells overexpress receptors for interleukin 13 and are    extremely sensitive to a novel chimeric protein composed of    interleukin 13 and pseudomonas exotoxin. Clin. Cancer Res. 1995    November; 1(11):1253-8.-   25. Madhankumar A B, Mintz A, Debinski W. Interleukin 13 mutants of    enhanced avidity toward the glioma-associated receptor, IL13Ralpha2.    Neoplasia. 2004 January; 6(1):15-22.-   26. Thompson J P, Debinski W. Mutants of interleukin 13 with altered    reactivity toward interleukin 13 receptors. J. Biol. Chem. 1999 Oct.    15; 274(42):29944-50.-   27. Debinski W, Gibo D M, Obiri N I, Kealiher A, Puri R K. Novel    anti-brain tumor cytotoxins specific for cancer cells. Nat.    Biotechnol. 1998 May; 16(5):449-53.-   28. Kalderon D, Roberts B L, Richardson W D, Smith A E. A short    amino acid sequence able to specify nuclear location. Cell 1984    December; 39(3 Pt 2):499-509.-   29. Hubner S, Xiao C Y, Jans D A. The protein kinase CK2 site    (Ser111/112) enhances recognition of the simian virus 40 large    T-antigen nuclear localization sequence by importin. J. Biol. Chem.    1997 Jul. 4; 272(27):17191-5.-   30. Rihs H P, Peters R. Nuclear transport kinetics depend on    phosphorylation-site-containing sequences flanking the karyophilic    signal of the Simian virus 40 T-antigen. EMBO J. 1989 May;    8(5):1479-84.-   31. Rihs H P, Jans D A, Fan H, Peters R. The rate of nuclear    cytoplasmic protein transport is determined by the casein kinase II    site flanking the nuclear localization sequence of the SV40    T-antigen. EMBO J. 1991 March; 10(3):633-9.-   32. Xiao C Y, Hubner S, Jans D A. SV40 large tumor antigen nuclear    import is regulated by the double-stranded DNA-dependent protein    kinase site (serine 120) flanking the nuclear localization    sequence. J. Biol. Chem. 1997 Aug. 29; 272(35):22191-8.-   33. Madhankumar A B, Mintz A, Debinski W. Alanine-scanning    mutagenesis of alpha-helix D segment of interleukin-13 reveals new    functionally important residues of the cytokine. J. Biol. Chem. 2002    Nov. 8; 277(45):43194-205.-   34. Mintz A, Gibo D M, Madhankumar A B, Debinski W. Molecular    targeting with recombinant cytotoxins of interleukin-13 receptor    alpha2-expressing glioma. J. Neurooncol. 2003 August;    64(1-2):117-23.-   35. Jans D A, Xiao C Y, Lam M H. Nuclear targeting signal    recognition: a key control point in nuclear transport? Bioessays    2000 June; 22(6):532-44.-   36. Costantini D L, Chan C, Cai Z, Vallis K A, Reilly R M.    (111)In-labeled trastuzumab (Herceptin) modified with nuclear    localization sequences (NLS): an Auger electron-emitting    radiotherapeutic agent for HER2/neu-amplified breast cancer. J.    Nucl. Med. 2007 August; 48(8):1357-68.-   37. Buchegger F, Perillo-Adamer F, Dupertuis Y M, Delaloye A B.    Auger radiation targeted into DNA: a therapy perspective. Eur. J.    Nucl. Med. Mol. Imaging 2006 November; 33(11):1352-63.-   38. Goddu S M, Rao D V, Howell R W. Multicellular dosimetry for    micrometastases: dependence of self-dose versus cross-dose to cell    nuclei on type and energy of radiation and subcellular distribution    of radionuclides. J. Nucl. Med. 1994 March; 35(3):521-30.-   39. Faraggi M, Gardin I, de Labriolle-Vaylet C, Moretti J L, Bok    B D. The influence of tracer localization on the electron dose rate    delivered to the cell nucleus. J. Nucl. Med. 1994 January;    35(1):113-9.-   40. Madhankumar A B, Slagle-Webb B, Wang X, Yang Q X, Antonetti D A,    Miller P A, Sheehan J M, Connor J R. Efficacy of interleukin-13    receptor-targeted liposomal doxorubicin in the intracranial brain    tumor model. Mol. Cancer Ther. 2009 March; 8(3):648-54.-   41. Liang H, Shin D S, Lee Y E, Nguyen D C, Trang T C, Pan A H,    Huang S L, Chong D H, Berns M W. Subcellular phototoxicity of    5-aminolaevulinic acid (ALA). Lasers Surg. Med. 1998; 22(1):14-24.-   42. Takemura T, Ohta N, Nakajima S, Sakata I. Critical importance of    the triplet lifetime of photosensitizer in photodynamic therapy of    tumor. Photochem. Photobiol. 1989 September; 50(3):339-44.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A compound comprising, in combination: an IL-13Rα2 binding ligand; atleast one effector molecule; a cytosol localization element covalentlycoupled between said binding ligand and said at least one effectormolecule; and a subcellular compartment localization signal elementcovalently coupled between said binding ligand and said at least oneeffector molecule.
 2. A compound of claim 1, wherein said compound hasthe formula, from N terminus to C terminus, selected from the groupconsisting of: A-B-C-D-E, E-D-C-B-A, A-B-D-C-E; and E-C-D-B-A, wherein:A is an IL-13Rα2 binding ligand; B is said cytosol localization element;C is said subcellular compartment localization signal element; D ispresent or absent and when present a first effector molecule; and E ispresent or absent and when present is a second effector molecule.
 3. Thecompound of claim 1, wherein said compound is a fusion protein orcovalent conjugate.
 4. The compound of claim 1, wherein each of A, B,and C, and optionally D and E, is a protein or peptide.
 5. The compoundof claim 1, wherein said cytosol localization element is a Pseudomonasexotoxin or diphtheria toxin translocation domain.
 6. The compound ofclaim 5, wherein said cytosol localization element is a Pseudomonasexotoxin A D2 segment.
 7. The compound of claim 1, wherein saidsubcellular compartment localization signal element is a nuclearlocalization element or a lysosomal localization element.
 8. Thecompound of claim 7, wherein said subcellular compartment localizationsignal element is an SV40 T antigen nuclear localization signal.
 9. Thecompound of claim 1, wherein said IL-13Rα2 binding ligand is IL-13, amutant of IL-13, or an IL-13Rα2 binding fragment thereof.
 10. Thecompound of claim 1, wherein said at least one effector moleculeconsists of two effector molecules.
 11. The compound of claim 1, whereinD is a toxin and E when present is an amphipathic antimicrobial peptide.12. The compound of claim 11, wherein said toxin is selected from thegroup consisting of diphtheria toxin and Pseudomonas exotoxin.
 13. Thecompound of claim 11, wherein said amphipathic antimicrobial peptidecomprises a sequence selected from the group consisting of: (KLAKLAK)₂(SEQ ID NO: 60); (KLAKKLA)₂ (SEQ ID NO: 61); (KAAKKAA)₂ (SEQ ID NO: 62)and (KLGKKLG)₂ (SEQ ID NO: 63).
 14. The compound of claim 1, whereinsaid at least one effector molecule comprises a therapeutic agent, adetectable group, a lipid, or a liposome.
 15. A nucleic acid thatencodes a compound of claim 1, and wherein said compound is a protein orpeptide.
 16. A host cell that contains a nucleic acid of claim 15 andexpresses the encoded peptide.
 17. A method of treating cancer in asubject in need thereof, comprising administering said subject a peptideof claim 1 in a treatment effective amount.
 18. The method of claim 17,wherein said cancer is selected from the group consisting of breastcancer, bladder cancer, pancreatic cancer, colorectal cancer, head andneck cancer, thyroid cancer, prostate cancer, and gliomas.
 19. Themethod of claim 18, wherein said cancer is glioblastoma multiforme. 20.A method of detecting IL-13Rα2 expressing cells, comprisingadministering a compound of claim 1 to a cell or group of cells anddetecting a detectable group coupled to said compound.
 21. A method ofdelivering at least one effector molecule to a subcellular compartmentof a cell of interest, comprising: contacting a compound of claim 1including at least one effector molecule to a cell of interest underconditions in which said compound is internalized therein and saideffector molecule is delivered to said subcellular compartment.
 22. Themethod of claim 21, wherein said subcellular compartment is the nucleus.23. The method of claim 22, wherein said at least one effector moleculecomprises a detectable group.
 24. The method of claim 21, wherein saidcompound further comprises an additional effector molecule (e.g., aseither D or E).
 25. The method of claim 24, wherein said additionaleffector molecules is delivered to the cytosol of said cell of interest(e.g., wherein said compound is of the formula A-B-D-C-E or E-C-D-B-A).